Long-term treatment of clarithromycin at a low concentration improves hydrogen peroxide-induced oxidant/antioxidant imbalance in human small airway epithelial cells by increasing Nrf2 mRNA expression

Background: Clarithromycin (CAM), a representative macrolide antibiotic, has been used widely at low doses for long-term therapy of chronic inflammatory airway diseases. Anti-inflammatory effects of macrolide antibiotics were first discovered in clinical practice. Although oxidative stress is known as a key pathogenesis factor in chronic airway inflammatory diseases, the mechanism of action of low-dose, long-term CAM therapy remains unclear. We aimed to examine the cytoprotective action of CAM against hydrogen peroxide (H2O2)-induced cell dysfunction, focusing on CAM dose and treatment duration, and using human small airway epithelial cells (SAECs), the main cells involved in chronic airway inflammatory diseases. Methods: SAECs were pretreated with CAM (1, 5 or 10 mu M) for 24, 48 or 72 h, and were subsequently exposed to H2O2 for 0.5-4 h. Levels of interleukin (IL)-8, glutathione (GSH) and glutathione disulfide (GSSG), and the activities of nuclear factor (NF)-.B and gamma-glutamylcysteine synthetase (gamma-GCS) were assayed using specific methods. IL-8 mRNA and NF erythroid 2-related factor 2 (Nrf2) mRNA expression were measured using real-time reverse transcription polymerase chain reaction (RT-PCR). Tukey's multiple comparison test was used for analysis of statistical significance. Results: Pretreatment with low-dose (1 or 5 mu M), long-term (72 h) CAM inhibited H2O2-induced IL-8 levels, NF-.B activity, and IL-8 mRNA expression, and improved the GSH/GSSG ratio via the maintenance of gamma-GCS expression levels. Similar to its enhancing effect on the GSH/GSSG ratio, pretreatment with low-dose CAM for 72 h significantly increased Nrf2 mRNA expression (p < 0.01 and p < 0.05). In contrast, these alterations were not observed after pretreatment with high-dose (10 mu M) or short-term (24 and 48 h) CAM. Conclusions: CAM is efficacious against cell dysfunction caused by oxidative stress under low-dose, long-term treatment conditions. This effect depended on the suppression of NF-kappa B activation and improvement of the H2O2-induced oxidant/antioxidant imbalance that is achieved by increasing Nrf2 mRNA expression in SAECs. The present study may provide the first evidence of why low-dose, long-term administration of macrolides is effective for treating chronic inflammatory airway diseases.